EP2904006A1 - Improved process for purifying lactoferrin from milk and products thereof - Google Patents
Improved process for purifying lactoferrin from milk and products thereofInfo
- Publication number
- EP2904006A1 EP2904006A1 EP13844731.3A EP13844731A EP2904006A1 EP 2904006 A1 EP2904006 A1 EP 2904006A1 EP 13844731 A EP13844731 A EP 13844731A EP 2904006 A1 EP2904006 A1 EP 2904006A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- lactoferrin
- milk
- rnases
- growth factors
- treatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- This invention relates generally to processes for purifying proteins of interest from milk.
- Milk from domestic animals has been used as a source of proteins and other products for the food and pharmaceutical industries for many years, and a variety of techniques are known for isolating these products.
- Milk is a colloidal suspension composed primarily of fats, lactose and proteins in water.
- milk contains an average of 30 to 140 grams of protein per litre, or about 4-17% by weight, depending on the species.
- the bulk of these proteins are caseins, which are complexed with calcium and phosphate in supramolecular structures known as micelles.
- the other major class of milk proteins is whey proteins, predominantly comprised of beta-lactoglobulin and alpha-lactalbumin, but also including lactoferrin, immunoglobulins, and serum albumin.
- Milk proteins usually are isolated by a combination of processes including membrane filtration techniques as well as ion exchange adsorption procedures.
- Lactoferrin is an 80 kD iron-binding glycoprotein found naturally in biological fluids such as saliva, bile, bronchial mucus, gastrointestinal fluids, cervico-vaginal mucus, seminal fluid, and milk.
- the richest source of lactoferrin is mammalian milk and colostrum.
- the concentration of lactoferrin in bovine skimmed milk is usually small, typlically between 80- 200 mg/ml depending on factors including the pasteurisation and other pre-treatment history of the skimmed milk. After precipitation of the casein present in milk the concentration of lactoferrin in bovine whey is typically 10-100 mg/ml depending on the physical and chemical pre-treatment of the whey.
- Lactoferrin has multiple postulated biological roles, including regulation of iron metabolism, immune function, and embryonic development. Lactoferrin has anti-microbial activity against a range of pathogens including Gram positive and Gram negative bacteria and fungi, including yeasts. The anti-microbial effect of lactoferrin is based on its capability of binding iron, which is essential for the growth of the pathogens. Lactoferrin also inhibits the replication of several viruses and increases the susceptibility of some bacteria to antibiotics and lysozyme by binding to the lipid A component of lipopolysaccharides on bacterial membranes. It is an aim of a preferred embodiment of the present invention to provide an improved method for purifying lactoferrin from milk, particularly bovine milk, to improve purity.
- a first aspect provides a process for purifying lactoferrin from milk, the process comprising subjecting the milk to filtration to separate it into a retentate fraction comprising lactoferrin and a permeate fraction comprising growth factors and/or RNAses, wherein prior to and/or during filtration the milk is subjected to salt treatment such that growth factors and/or RNAses flow into the permeate.
- the inventors When purifying lactoferrin from milk using membrane filtration with a 30 kD or 50 kD cut off the inventors found that the lactoferrin retentate was contaminated with growth factors and RNAses. As these growth factors and RNAses have a molecular weight of less than 30 kD they should have passed through the membrane and into the permeate and not be present as an impurity in the retentate lactoferrin fraction. Accordingly the discovery of growth factors and RNAses in the retentate was surprising. With the aim of further purifying the lactoferrin fraction the inventors found that the addition of a large amount of salt to the milk prior to filtration removed the growth factors and RNAses from the retentate, thus further purifying the lactoferrin. Under salt conditions the growth factors and RNAses were found in the permeate.
- the inventors propose that under normal conditions the RNAses and growth factors in milk aggregate or otherwise form a mass that is larger than their individual molecular weights. It is proposed that the salt treatment causes the RNAses and growth factors to disaggregate or disassociate.
- the process of the first aspect provides a process for purifying lactoferrin from milk the process comprising subjecting the milk to filtration to separate it into a retentate fraction comprising lactoferrin and a permeate fraction comprising growth factors and/or RNAses, wherein prior to and/or during filtration the milk is subjected to salt treatment capable of disaggregating or disassociating any mass of RNAses or growth factors so that growth factors and/or RNAses flow into the permeate.
- the inventors alternatively propose that under conditions of low ionic strength, protein aggregates may become associated with the membrane, thereby forming a layer with a smaller apparent pore size to that of the membrane, which prevents RNAses and growth factors in milk passing through the membrane. It is proposed that the salt treatment prevents formation of or removes the protein layer, allowing any RNAses and growth factors to pass through the membrane into the permeate.
- the process of the first aspect provides a process for purifying lactoferrin from milk the process comprising subjecting the milk to membrane filtration to separate the milk into a retentate fraction comprising lactoferrin and a permeate fraction comprising growth factors and/or RNAses, wherein prior to and/or during filtration the milk is subjected to salt treatment capable of separating any RNAses or growth factors from the membrane such that growth factors and/or RNAses flow into the permeate.
- a second aspect provides lactoferrin obtained from the process of the first aspect.
- the lactoferrin of the second aspect is subjected to further purification.
- a third aspect provides the use of lactoferrin of the second aspect for as an anticancer, anti-viral, anti-microbial or anti-inflammatory agent, for treating proliferative disorders such including cancer, particularly those involving solid tumours, for treating malignant neoplasms and hyperproliferative diseases, for treating bone disorders by promoting osteogenesis, for treating viral or microbial infections, for corneal wound treatment, to boost immunity, to treat B-cell non-Hodgkin lymphoma, to treat septic shock, to improve gut health by improving bifidus numbers while decreasing E.coli, streptococcus and Clostridium, to treat IBD, for promoting immunity and resistance to disease, to manage systemic inflammation, to prevent organ transplant rejection and graft versus host disease, to improve maternal and fetal health, to improve neuronal health and infant gut development, to reduce the likelihood of pregnancy-associated complications such as pre-eclampsia, preterm labour, gestational diabetes, miscarriage, intrauterine fetal death, premature
- the third aspect provides lactoferrin obtained from the process of the first aspect for use as proposed in the third aspect or for use in the manufacture of a medicament for use as proposed in the third aspect.
- Figure 1 shows permeate flux across a 50 kDa membrane increases as conductivity increases.
- FIG 2 shows ultrafiltration in the presence of increasing sodium chloride increases the purity of retained lactoferrin by increasing the permeation of low molecular weight contaminants (RNAses used as an example).
- RNAses low molecular weight contaminants
- Figure 3 shows the concentration of lactoferrin (*), low molecular weight proteins (RNAses used as an example, ) and protein (A) in the permeate from a 50 kDa UF membrane.
- Figure 4 A,B,C and D show size exclusion HPLC analysis of lactoferrin in increasing concentrations of sodium chloride, showing that increasing concentrations disaggregate large particles.
- the sodium chloride concentration within the first 50 minutes is the important difference between the four chromatographs: A, 0 M NaCI; B, 0.16 M NaCI; C, 0.32 M NaCI and D, 0.48 M NaCI.
- Figures 5A and B show ultrafiltration of a lactoferrin solution through a 50 kDa membrane held in a Vivacell apparatus, in the presence of sodium chloride (0, 1.5, 3, 4,5, or 6% NaCI), showing that in the presence of sodium chloride, the 50 kDa membrane selectively allows the transmission of RNAses and other growth factors.
- Figures 6A and 6B shows ultrafiltration of a lactoferrin solution through a 50 kDa membrane held in a Vivacell apparatus, in the presence of sodium chloride (0, 1.5, 3, 4,5, or 6% NaCI), showing that in the presence of sodium chloride, the 50 kDa membrane selectively allows the transmission of RNAses and other growth factors.
- Figure 7 shows ultrafiltration in the presence of increasing conductivity increases the purity of retained lactoferrin.
- Figure 8 shows that the increased lactoferrin purity observed is due to the increased transmission of contaminating growth factors, such as the RNAses. Growth factor transmission increases quickly between 0 mS and 20 mS.
- Figure 9 shows lactoferrin purity in the lactoferrin starting material, main cation exchange chromatography peak and the material further purified by 50 kDa UF.
- Target lactoferrin purity is 98%.
- Figure 10 illustrates the increase in lactoferrin purity during processing with 50 kDa membranes. Samples analysed by cation HPLC in the liquid form are indicated in grey and samples analysed by cation exchange HPLC in the powder form are indicated in black.
- Figure 11 illustrates that when lactoferrin solutions containing sodium chloride are processed by ultrafiltration, increasing membrane molecular weight cut off allows an increasing amount of protein to pass into the permeate ( J A J) and improves the retention of protein ( * ) and lactoferrin ( ). Results have been obtained by testing separating 2 mg/mL lactoferrin solution containing 6 % sodium chloride with Sartorius Stedium Vivacell 250 UF membranes of the indicated porosities.
- Figure 12 illustratyes that flux of membranes increases with molecular weight cut off (data obtain from Synder Filtration, water processed at 25°C and 3.4 Bar), but is not appreciably different between 30 kDa and 50 kDa.
- Figure 13 illustrates that when lactoferrin solutions containing sodium chloride are processed by ultrafiltration, increasing membrane molecular weight cut off increases the purity until the membrane porosity is 50 kDa. Results have been obtained by testing separating 2 mg/mL lactoferrin solution containing 6 % sodium chloride with Sartorius Stedium Vivacell 250 UF membranes of the indicated porosities.
- Figure 14 illustrates the increase in lactoferrin purity due to processing through UF7 fitted with either the traditional 5 kDa membranes or the new 50 kDa membrane. Plotted values are means and bars indicate 95% confidence interval.
- the present invention provides improved methods for purifying lactoferrin from milk or enriching milk for lactoferrin.
- the inventors have recognised the need for a process which allows the preparation of enriched lactoferrin in an efficient manner.
- the terms "purified” or "enriched” as used herein in relation to lactoferrin means that the lactoferrin protein:total protein ratio present in the retentate is increased relative to the ratio present in the milk before the filtration step.
- the fraction to be considered enriched or purified it should have a lactoferrin content of at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 27, 28, or 30, 40, 50, 60, 70, 80, 90, 95, 96, 97, 98, 98, or 99% w/w higher than in milk before the filtration step.
- the process of the first aspect seeks to increase the purity of the lactoferrin retentate to up to 100% pure.
- fraction refers to a partially purified portion of milk.
- milk includes whole milk, skim milk, buttermilk, whey (such as acid or cheese/renneted whey) or a whey derivative (such as whey protein concentrate or whey protein isolate flow through), and colostrum. It also includes milk fractions, for example fractions that have been subjected to purification steps such as cation exchange
- Such fractions include milk basic protein and enriched lactoferrin fractions.
- the milk may be obtained from any lactating animal, e.g. ruminants such as cows, sheep, buffalos, goats, and deer, non- ruminants including primates such as a human, and monogastrics such as pigs. It is preferred that skim milk which is derived from whole cow's milk is used in the process of the present invention.
- lactating animal e.g. ruminants such as cows, sheep, buffalos, goats, and deer
- non- ruminants including primates such as a human
- monogastrics such as pigs.
- skim milk which is derived from whole cow's milk is used in the process of the present invention.
- the filtration used in the process of the first aspect comprises membrane filtration.
- the membrane has a size cut off of 25 kD, 30 kD, 35 kD, 40 kD, 45 kD, 50 kD, 55 kD, 60 kD, 65 kD, 70 kD or 75 kD.
- the cut off is preferably 30 kD or over and 50 kD or less.
- the filtration may involve ultrafiltration or diafiltration or both.
- the salt treatment used in the process of the first aspect involves adding sufficient salt so that the ionic strength of the milk is at least 0.2 M (1.1 %) NaCI or equivalent.
- the ionic strength is maintained at least this level for a period required to obtain the required increase in lactoferrin purity. This period may be at least 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 1 hours, 12 hours or more depending on the milk feed material and the increase in purity desired.
- the salt treatment used in the method of the first aspect involves adding sufficient salt so that the conductivity of the milk is at least 20 mS.
- the conductivity is maintained at at least this level during the entire filtration step.
- the milk will have an ionic strength of substantially less than 0.2M NaCI or equivalent or less that 20 mS conductivity and hence the salt treatment involves the addition of salt to increase the ionic strength or conductivity of the milk to the desired level.
- the milk will have an ionic strength that may be at least 0.2 M NaCI or equivalent or the conductivity will be 20 mS or more.
- fractions from cation exchange are subjected to treatment to remove salt (e.g. diafiltration with water).
- the salt treatment is such to maintain the ionic strength of the milk at at least 0.2 M (1.1 %) NaCI or equivalent during the filtration step or to maintain the conductivity of the milk at at least 20mS during the filtration step.
- conductivity is the ability of a material to conduct electric current. Conductivity is generally measured using a conductivity meter for example a Hach Sension 5. Persons skilled in the art would be aware of suitable alternative means to measure conductivity. There is a generally linear relationship between sodium ion concentration and conductivity.
- the salt used in the salt treatment is not limited and alternatives to NaCI would be known to the person skilled in the art.
- any soluble, non-toxic buffer can be used such as the soluble sodium, potassium, calcium, magnesium or lithium salts of chloride, citrate, phosphate, acetate, sulphate, bicarbonate, hydroxide, imidazole, or maleate.
- Synthetic zwitterion buffers such as Trizma, HEPES or tricine may also be used.
- the ionic strength of the milk must be at least 0.2 M (1.1 %) NaCI or equivalent or more prior to the filtration step.
- the ionic strength of the milk is increased by adding 1.1 %, salt, 1.5% salt, 2% salt, 2.5% salt, 3% salt, 3.5% salt, 4% salt, 4.5% salt, 5% salt, 5.5% salt, 6% salt or more.
- the ionic strength of the milk is increased to 0.2M,
- the conductivity is 20 mS, 30 mS, 40 mS, 50 mS, 60 mS, 70 mS. 80 mS, 90 mS, 100 mS, 110 mS, 120 mS or more.
- the salt treatment involves the addition of 0.2-0.5M NaCI or KCI to the milk prior to the filtration step.
- the salt treatment is carried out at 4-10 degrees.
- the salt treatment is carried out at 10-30 degrees.
- the salt treatment is carried out at 30-50 degrees.
- the salt treatment is carried out at 50-70 degrees. In one embodiment the salt treatment is carried out at less than 20 degrees.
- the salt treatment is carried out at 50 degrees or higher as at temperatures in excess of 50 degrees coliform formation is reduced.
- lactoferrin denatures at roughly 70 degrees performing the salt treatment at 60 degrees or above may result in decreased yield.
- the filtration step is carried out at 4-10 degrees.
- the filtration step is carried out at 10-30 degrees.
- the filtration step is carried out at 30-50 degrees.
- the filtration step is carried out at 50-70 degrees.
- the salt treatment is carried out at atmospheric pressure.
- the filtration step is carried out at a transmembrane pressure less than 2.5 Bar per membrane, more likely less than 2.0 Bar, more likely again less than 1.5 Bar but ideally 1.0-1.4 Bar, although a pressure of 0.0-1.0 Bar could also work acceptably but have a lower transmembrane flux.
- Treating refers to both therapeutic treatment and prophylactic or preventative measures, wherein the aim is to prevent, ameliorate, reduce or slow down (lessen) or improve a condition, disease or disorder.
- Treating covers any treatment of, or prevention of a condition in a vertebrate, a mammal, particularly a human.
- Preventing refers to keeping from occurring, or to hinder, defend from, or protect from the occurrence of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
- a subject in need of prevention may be prone to develop the condition.
- ameliorate or “amelioration” refers to a decrease, reduction or elimination of a condition, disease, disorder, or phenotype, including an abnormality or symptom.
- a subject in need of treatment may already have the condition, or may be prone to have the condition or may be in whom the condition is to be prevented.
- maintain refers to sustaining a condition at pre-treatment levels.
- the lactoferrin of the second aspect may be provided as a pharmaceutical, veterinary or nutraceutical composition or as a food.
- a pharmaceutical composition is one which is suitable for administration to humans.
- a veterinary composition is one that is suitable for administration to animals. Generally such compositions will contain purified lactoferrin or at the very least all components of the composition will be verifiable.
- the pharmaceutical or veterinary composition may comprise one or more carriers and optionally other therapeutic agents. Each carrier, diluent, adjuvant and/or excipient may be "acceptable”.
- acceptable is meant a material which is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected active agent without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical or veterinary composition in which it is contained.
- a “acceptable” salt or ester of a novel compound as provided herein is a salt or ester which is not biologically or otherwise undesirable.
- a “carrier” is an acceptable solvent, suspending agent or vehicle for delivering the agent to the subject.
- the carrier may be liquid or solid and is selected with the planned manner of administration in mind.
- Each carrier must be "acceptable” in the sense of being not biologically or otherwise undesirable i.e. the carrier may be administered to a subject along with the agent without causing any or a substantial adverse reaction.
- the pharmaceutical or veterinary composition may be administered orally, topically, or parenterally in formulations containing conventional non-toxic acceptable carriers, adjuvants, and vehicles.
- parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, subconjunctival, intracavity, transdermal and subcutaneous injection, aerosol for administration to lungs or nasal cavity or administration by infusion by, for example, osmotic pump.
- the pharmaceutical or veterinary composition may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs.
- the composition for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations.
- Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable
- disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, xanthan gum, bentonite, alginic acid or agar.
- Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring.
- Suitable preservatives include sodium benzoate, vitamin E, alphatocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite.
- Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc.
- Suitable time delay agents include glyceryl monostearate or glyceryl distearate.
- the tablets may contain the agent in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
- excipients may be, for example, (1) inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; (2) granulating and disintegrating agents, such as corn starch or alginic acid; (3) binding agents, such as starch, gelatin or acacia; and (4) lubricating agents, such as magnesium stearate, stearic acid or talc.
- These tablets may be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
- a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
- Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
- non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
- Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
- Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride
- lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
- Preservatives and other additives may also be present such as, for example, anti-microbials, anti-oxidants, chelating agents, growth factors and inert gases and the like.
- the pharmaceutical or veterinary composition may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions.
- the pharmaceutical or veterinary composition may also be included in a container, pack, or dispenser together with instructions for administration.
- the pharmaceutical or veterinary composition can be administered in one dose, or at intervals such as once daily, once weekly, and once monthly.
- Dosage schedules can be adjusted depending on the half life of the active agent, or the severity of the subject's condition.
- the pharmaceutical or veterinary composition is administered as a bolus dose, to maximize the circulating levels of active agent for the greatest length of time after the dose. Continuous infusion may also be used after the bolus dose.
- the lactoferrin of the second aspect may be provided in a nutraceutical composition or food.
- nutraceutical refers to an edible product isolated or purified from food, in this case from a milk product, which is demonstrated to have a physiological benefit or to provide protection or attenuation of an acute or chronic disease or injury when orally administered.
- the nutraceutical may thus be presented in the form of a dietary preparation or supplement, either alone or admixed with edible foods or drinks.
- the nutraceutical composition may be in the form of a soluble powder, a liquid or a ready-to-drink formulation.
- the nutritional composition may be in solid form as a food; for example in the form of a ready-to-eat bar or breakfast cereal.
- Various flavours, fibres, sweeteners, and other additives may also be present.
- the nutraceutical preferably has acceptable sensory properties (such as acceptable smell, taste and palatability), and may further comprise vitamins and/or minerals selected from at least one of vitamins A, B1 , B2, B3, B5, B6, B11 , B12, biotin, C, D, E, H and K and calcium, magnesium, potassium, zinc and iron.
- acceptable sensory properties such as acceptable smell, taste and palatability
- vitamins and/or minerals selected from at least one of vitamins A, B1 , B2, B3, B5, B6, B11 , B12, biotin, C, D, E, H and K and calcium, magnesium, potassium, zinc and iron.
- the nutraceutical composition may be produced as is conventional; for example, the composition may be prepared by blending together the protein and other additives. If used, an emulsifier may be included in the blend. Additional vitamins and minerals may be added at this point but are usually added later to avoid thermal degradation.
- the protein may be admixed with additional components in powdered form.
- the powder should have a moisture content of less than about 5% by weight.
- Water preferably water which has been subjected to reverse osmosis, may then be mixed in to form a liquid mixture.
- the nutraceutical composition is to be provided in a ready to consume liquid form, it may be heated in order to reduce the bacterial load. If it is desired to produce a liquid nutraceutical composition, the liquid mixture is preferably aseptically filled into suitable containers. Aseptic filling of the containers may be carried out using techniques commonly available in the art. Suitable apparatus for carrying out aseptic filling of this nature is commercially available.
- nutraceutical composition also comprises one or more
- Nutraceutical compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans; mannitol; proteins;
- polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA; adjuvants and preservatives.
- the nutraceutical may be an infant formula, particularly a humanised milk formula for administration to infants.
- the lactoferrin of the second aspect can take the form of a food supplement, a nutritional formulation, a sports nutrition supplement or an infant formula.
- the food is animal feed.
- Example 1 Ultrafiltration of lactoferrin to remove small molecules and increase lactoferrin purity
- Retentate was recycled to the feed tank and permeate was collected.
- the permeate removed was replaced with diafiltration solution of the same conductivity. Purity was assessed by cation exchange HPLC.
- the lactoferrin solution eluted from chromatographic plants and collected in UF4 is intended to be 96 mS, so the optimum increase in lactoferrin purity occurring in the region between 80 mS and 100 mS is compatible with the existing process without further processing.
- Lactoferrin (95% lactoferrin as percentage of protein) was dissolved in water at 10 mg/mL.
- a HPLC was fitted with a Superdex 200 10/300 GL column (GE Healthcare) and equilibrated with Buffer A (Tris.HCI (pH 7, 1 g/L)) at a flow of 0.5 mL/min.
- Buffer B is Tris.HCI (1 g/L) containing 1 M sodium chloride (pH 7) and the sample injected was 100 ⁇ .
- the amount of Buffer B was 0 % between 0 min to 50 min, increased to 100% buffer B between 50 min and 90 min and then increased to 16% Buffer B from 90 min to 150 min.
- the salt between 0 min to 50 min and 90 min to 150 min increased by 16 % B.
- the lactoferrin elutes in a single major peak at approximately 28 min and is followed by several smaller peaks, which include lactoferrin fragments and non-lactoferrin proteins.
- Chromatograph D shows, which contained 0.48M NaCI, is an example of a typical lactoferrin profile.
- Chromatograph A which contained no NaCI, showed the presence of a smaller lactoferrin peak and a larger peak at 94 min.
- the peak at 94 min is lactoferrin that was insoluble until the higher salt commencing at 50 min dissociated the aggregated lactoferrin and allowed it to move through the resin bed contained in the column.
- Example 3 Ultrafiltration of lactoferrin to remove small molecules and increase lactoferrin purity
- Vivacell 250 units (Sartorius) were fitted with 50 kDa membranes. Each was filled to the 250 mL line with 1 mg/mL lactoferrin solution, but each was had a different sodium chloride concentration (0, 1.5, 3, 4, 5 or 6 % NaCI). The cells were pressurised to 3 Bar. Samples of retentate and permeate were collected and analysed by cation exchange HPLC. It was found that increasing salt concentration increased the transmission of growth factors (RNAses used as a representative) and increased the purity of the lactoferrin in the retentate ( Figures 5 and 6).
- Example 4 Ultrafiltration of lactoferrin to remove small molecules and increase lactoferrin purity
- Lactoferrin purity was increased from 90.9% (0 mS) to 93.5% (60 mS) ( Figure 7), which represents a 2.5% increase in purity.
- the increase in lactoferrin purity was achieved by the selective removal of growth factors (RNAses used as an example, Figure 8).
- Example 5 Using secondary cation exchange chromatography with or without ultrafiltration to improve increase lactoferrin purity
- Lactoferrin powder (40 g, 95.2 % lactoferrin) was dissolved in water (200mL) with a Stomacher. A sample was collected and analysed by cation exchange HPLC. The lactoferrin solution was applied to a column (50 mmD ⁇ 1000 mmL) filled with SP Fast Flow resin (GE Healthcare). The bound lactoferrin was eluted from the resin with a linear gradient of 3 L, ranging from 0 % to 5.8 % (1 M) sodium chloride. The main lactoferrin peak was collected and analysed by cation exchange HPLC.
- Lactoferrin powder (1 kg, purity 96.3 % protein) was dissolved in 100 L water.
- the lactoferrin solution was placed in an APV UF plant fitted with a single Synder 6.3" 50 kDa membrane (BX-5XB-6338).
- the combined volume of the lactoferrin solution and the water present in the UF plant was 180 L and the conductivity was 58 mS due to added sodium chloride.
- Permeate (100 L) was removed in 10 min. Water (100 L) and salt were added to obtain a conductivity of 121 mS. Permeate (100 L) was removed in 10 min.
- the conductivity of the lactoferrin retentate was then reduced to 1300 by water diafiltration (400L permeate).
- the lactoferrin retentate was then freeze-dried (48 h, 1 mBar, 40°C). Liquid samples were analysed at MGC by cation exchange HPLC and powder samples were analysed by cation exchange HPLC by an external laboratory (samples analysed in the powder form should be considered more accurate).
- lactoferrin The purity of lactoferrin can be increased by approximately 2 % (protein basis) when sufficient diafiltration has occurred in an UF plant fitted with 50 kDa membranes.
- the final purity result was 98.1 % (protein basis) ( Figure 10), which shows that the process may be viable and further development is warranted.
- Membrane molecular weight cut off is the most important influence on membrane performance. As the pore size of the membrane increases, the membrane allows the passage of larger proteins (Figure 1 1) and the flux increases (Figure 12). Membranes have the capacity to separate components in mixtures, but only if one or some components are smaller than the membrane MWCO and one or some components are larger than the membrane MWCO. In general, a difference in molecular weight of at least two orders of magnitude are required for ideal separations (ie 28 Da water and 58 Da sodium chloride can be almost completely separated from 78 kDa lactoferrin), but some separation is possible where such a significant differential in size is not present. If the aim is to increase the purity of lactoferrin solution, the 50 kDa membrane appears to be the best option available ( Figure 13).
- Example 8 Ultrafiltration membranes
- Spiral wound polyethersulfone (PES) MWCO 30 kDa
- the ultrafiltration plant known as UF4 was used to:
- PES is an inherently hydrophilic membrane that wets out quickly and completely resulting in fast filtration with superior flow rates and high throughputs. PES membrane is also extremely low protein binding minimizing the likelihood of target protein binding, which means high yields, stable transmembrane fluxes and consistent apparent membrane porosities.
- Type Spiral wound polyether sulphone (PES) MWCO: 5 kDa
- -5 kDa membranes retain the non-lactoferrin impurities, which prevents them from returning to the chromatographic process and contaminating the lactoferrin during subsequent lactoferrin elutions.
- a smaller membrane is required because the proteins mixture contains many smaller proteins, many of which are growth factors.
- Type Ceramic tubes (alumina) MWCO: 0.8 ⁇
- the microfiltration plant known as MF6 was used to:
- -ceramic membranes have a high tolerance for cleaning chemicals and can be aggressively cleaned to ensure the complete removal of microbes. They are also very robust membranes that do not tear, which may allow contaminating material to pass through the membrane. Justification of MWCO:
- the plant is 0.8 ⁇ , rather than 0.22 ⁇ , to allow the protein to be passed through the membrane at a reasonable volume and solids concentration.
- the filter is not intended to be a complete microbial removal step, since the main raw material (skimmed milk) has been previously pasteurised.
- PVDF polyvinylidene fluoride
- PVDF polyvinylidene fluoride
- -50 kDa membranes are used in preference to smaller membranes (historically 5 kDa membranes) because they allow improved transmission of non-lactoferrin proteins (RNases, growth factors), while retaining lactoferrin.
- RNases non-lactoferrin proteins
- the increased transmission of non-lactoferrin protein results in a higher lactoferrin purity (average increase in purity 1.8% protein, P ⁇ 0.001). Further details can be obtained from 'Increasing Lactoferrin Purity by Diafiltration with Salt Solution in an Ultrafiltration Plant Fitted with 50 kDa Membranes' (JR0010).
- lactoferrin 80 kDa is and for this reason lactoferrin eluting from the column at a low concentration (3% protein) can be concentrated to >20% protein, without a matching increase in the salt concentration or loss of protein.
- Example 9 Fully commercial scale trial to confirm that the purity of lactoferrin can be increased by 50 kDa UF
- a batch from the chromatographic process was split in half. Half was processed through UF7 fitted with 5 kDa membranes with no salt diafiltration and the other half was processed through UF7 fitted with 50 kDa membranes and salt diafiltration.
- the routine commercial scale process was modified by replacing the 5 kDa membranes present in UF7 with Synder 50 kDa membranes identical to those used in experiments 1 and 6.
- a salt diafiltration step was also added to the commercial process.
- the salt used in the diafiltration solution was prepared by adding 150 kg sodium chloride to 3000 kg water.
- Table 1 A comparison of the composition lactoferrin made through the standard UF7 process and the high purity UF7 process, showing that the powders are similar, except for the increase in lactoferrin purity.
- the commercial scale trial produced a powder with a lactoferrin purity of 98.0 % (protein basis), which was 1.1 % higher in purity than the UF4 solution from which it was derived.
- the process was again shown to work successfully at a fully commercial scale.
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AU2012904391A AU2012904391A0 (en) | 2012-10-08 | Improved process for purifying milk proteins and products thereof | |
AU2013204858A AU2013204858B2 (en) | 2012-10-08 | 2013-04-12 | Improved process for purifying milk proteins and products thereof |
PCT/AU2013/001152 WO2014056025A1 (en) | 2012-10-08 | 2013-10-08 | Improved process for purifying lactoferrin from milk and products thereof |
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EP2904006A1 true EP2904006A1 (en) | 2015-08-12 |
EP2904006A4 EP2904006A4 (en) | 2016-04-06 |
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US (1) | US10016487B2 (en) |
EP (1) | EP2904006A4 (en) |
JP (1) | JP6475163B2 (en) |
KR (1) | KR20150068404A (en) |
CN (1) | CN104768971B (en) |
AU (1) | AU2013204858B2 (en) |
BR (1) | BR112015007674A2 (en) |
CA (1) | CA2886892A1 (en) |
IN (1) | IN2015DN02971A (en) |
NZ (1) | NZ631826A (en) |
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AU2013204858B2 (en) | 2012-10-08 | 2015-06-25 | Saputo Dairy Australia Pty Limited | Improved process for purifying milk proteins and products thereof |
CN106215174A (en) * | 2016-08-04 | 2016-12-14 | 无锡科捷诺生物科技有限责任公司 | A kind of formulation preparation method of human lactoferrin |
JP7370224B2 (en) * | 2019-11-08 | 2023-10-27 | ライオン株式会社 | Enteric-coated preparation containing lactoferrin |
FR3115037A1 (en) * | 2020-10-12 | 2022-04-15 | Compagnie Laitiere Europeenne | Process for purification of cationic protein fraction and fraction thus obtained |
CN113278063A (en) * | 2021-05-13 | 2021-08-20 | 上海仁统生物科技中心 | Method for obtaining lactoferrin from colostrum by ultrafiltration |
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EP0418704A1 (en) | 1989-09-22 | 1991-03-27 | Snow Brand Milk Products Co., Ltd. | Process of separating, purifying and recovering milk proteins capable of binding iron |
WO2014056025A1 (en) | 2012-10-08 | 2014-04-17 | Murray Goulburn Co-Operative Co. Limited | Improved process for purifying lactoferrin from milk and products thereof |
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FR2584727B1 (en) * | 1985-07-11 | 1988-06-17 | Roussel Uclaf | PROCESS FOR EXTRACTING MILK PROTEINS, PRODUCTS, APPLICATION OF THE PROCESS, AND PHARMACEUTICAL COMPOSITIONS |
US5221734A (en) | 1987-10-01 | 1993-06-22 | Ciba-Geigy Corporation | Process for preparing a polypeptide growth factor for milk |
GB8729031D0 (en) * | 1987-12-11 | 1988-01-27 | Express Foods Group Ltd | Isolation of immunoglobulin rich fraction from whey |
EP0620709B1 (en) * | 1992-01-15 | 1995-10-18 | Campina Melkunie B.V. | Process for isolating lactoferrin and lactoperoxidase from milk and milk products |
JPH05202098A (en) | 1992-01-29 | 1993-08-10 | Snow Brand Milk Prod Co Ltd | Production of physiologically active substance from lactic material |
US5756680A (en) * | 1994-01-05 | 1998-05-26 | Sepragen Corporation | Sequential separation of whey proteins and formulations thereof |
JPH09509165A (en) * | 1994-02-16 | 1997-09-16 | ファーミング ベスローテン フェンノートシャップ | Separation of lactoferrin from milk |
NL1005677C2 (en) * | 1997-03-27 | 1998-09-29 | Campina Melkunie Bv | Method for recovering growth factors, or a composition containing one or more growth factors, from milk or a derivative thereof. |
ATE420108T1 (en) * | 2001-07-20 | 2009-01-15 | Campina Bv | METHOD FOR PRODUCING PREPARATIONS FROM DAIRY PRODUCTS CONTAINING GROWTH FACTORS (TGF-BETA AND IGF-1), LACTOPEROXIDASE AND IMMUNOGLOBULINS WITH LOW MUTUAL IMPURITY |
US6875459B2 (en) * | 2001-09-10 | 2005-04-05 | Henry B. Kopf | Method and apparatus for separation of milk, colostrum, and whey |
NZ535784A (en) * | 2002-03-07 | 2006-02-24 | Upfront Chromatography As | A process of isolating lactoferrin |
FR2841747B1 (en) * | 2002-07-02 | 2004-08-20 | Cie Laitiere Europeenne | MILK PROTEIN ISOLATE AND PROCESS FOR ITS PREPARATION |
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EP0418704A1 (en) | 1989-09-22 | 1991-03-27 | Snow Brand Milk Products Co., Ltd. | Process of separating, purifying and recovering milk proteins capable of binding iron |
WO2014056025A1 (en) | 2012-10-08 | 2014-04-17 | Murray Goulburn Co-Operative Co. Limited | Improved process for purifying lactoferrin from milk and products thereof |
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KR20150068404A (en) | 2015-06-19 |
AU2013204858B2 (en) | 2015-06-25 |
US20150315264A1 (en) | 2015-11-05 |
AU2013204858A1 (en) | 2014-04-24 |
IN2015DN02971A (en) | 2015-09-18 |
US10016487B2 (en) | 2018-07-10 |
NZ631826A (en) | 2017-08-25 |
CA2886892A1 (en) | 2014-04-17 |
CN104768971A (en) | 2015-07-08 |
BR112015007674A2 (en) | 2017-07-04 |
RU2683228C2 (en) | 2019-03-26 |
JP2015536905A (en) | 2015-12-24 |
CN104768971B (en) | 2017-12-05 |
RU2015116933A (en) | 2016-11-27 |
WO2014056025A1 (en) | 2014-04-17 |
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